Generation of herpes simplex virus (HSV) mutants often requires generation of a unique plasmid by cloning an entire expression cassette consisting of a promoter, gene of interest and polyadenylation sequences into a plasmid separately constructed to contain the relevant flanking sequences and then co-transfecting BHK cells with the resultant plasmid and HSV-1 DNA. Homologous recombination drives the formation of recombinant HSV-1 expressing the gene of interest, which is identified by Southern blotting. The recombinant virus is plaque purified between 6-10 times by Southern blotting, dependent on the efficiency of the homologous recombination. This process can take between 3-6 months.
This approach was taken by Liu et al1 in generating two distinct plasmids, the first consisting of HSV-1 strain 17+ Sau3A fragment derived sequences flanking an expression cassette consisting of a CytoMegalovirus (CMV) promoter, Green Fluorescent Protein (GFP) gene and bGH polyadenylation (polyA) signal and the second wherein the GFP gene is replaced with either a human or mouse Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) gene.
Shuttle vectors have been used to generate recombinant adenoviral vectors, e.g. the pAdEasy™ system of vectors (Stratagene), for use in overexpressing recombinant proteins in mammalian cells. However, these vectors require the cloning of the gene of interest into a first shuttle vector which is then co-transformed into a specially constructed cell line to generate a recombinant adenoviral plasmid which is transfected into a separate specially constructed mammalian cell line in which the recombinant adenoviral plasmid is directly packaged into virus particles.
Terada et al2 sought to introduce therapeutic transgenes into an oncolytic HSV vector backbone by creating an HSV-BAC (bacterial artificial chromosome) carrying the entire MGH1 genome and a replication-conditional shuttle plasmid.
Creation of viral vectors, particularly HSV, which may encode a gene of interest capable of being expressed from the vector, is presently a slow and often complicated and inefficient process.
The HSV genome comprises two covalently linked segments, designated long (L) and short (S). Each segment contains a unique sequence flanked by a pair of inverted terminal repeat sequences. The long repeat (RL or RL) and the short repeat (RS or RS) are distinct.
The HSV ICP34.5 (also γ34.5 or RL1) gene, which has been extensively studied, has been sequenced in HSV-1 strains F and syn17+ and in HSV-2 strain HG52. One copy of the ICP34.5 gene is located within each of the RL repeat regions. Mutants inactivating both copies of the ICP34.5 gene (i.e. null mutants), e.g. HSV-1 strain 17 mutant 1716 (HSV1716) or the mutants R3616 or R4009 in strain F, are known to lack neurovirulence, i.e. be avirulent, and have utility as both gene delivery vectors or in the treatment of tumours by oncolysis. HSV1716 has a 759 bp deletion in each copy of the ICP34.5 gene located within the BamHI s restriction fragment of each RL repeat.
ICP34.5 null mutants such as HSV1716 are, in effect, first-generation oncolytic viruses. Most tumours exhibit individual characteristics and the ability of a broad spectrum first generation oncolytic virus to replicate in or provide an effective treatment for all tumour types is not guaranteed.
The prior art provides technically challenging, procedurally slow and inefficient materials and methods for generating recombinant HSV. In particular the prior art does not provide methods of, and materials for, generating recombinant HSV which are easy to detect, may be designed to be specific null mutants and which may express a selected gene of interest.
First generation oncolytic viruses such as HSV-1 strain 17 mutant 1716 show significant therapeutic potential in tumour and gene therapy. Overcoming the existing technical difficulties by enabling rapid generation and screening of second generation oncolytic viruses of this kind provides a significant improvement in the development of novel pharmaceutical compositions, vaccines and medicaments for the treatment of cancer and disease.
HSV 1716 is described in EP 0571410 and WO 92/13943 and has been deposited on 28 Jan. 1992 at the European Collection of Animal Cell Cultures, Vaccine Research and Production Laboratories, Public Health Laboratory Services, Portion Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number V92012803 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the ‘Budapest Treaty’).
The HSV-1 strain 17+ mutant 1716 lacks a functional ICP34.5 gene resulting in greatly reduced lethality in mice but replicates as wild-type virus in actively dividing tissue culture cells (MacLean et al 1991, Brown et al 1994). The ICP34.5 ORF is a neurovirulence gene and its protein product has been proposed to condition post-mitotic cells for viral replication, probably via an interaction with proliferating cell nuclear antigen (Brown et al 1997, Harland et al 2003). ICP34.5 deletion mutants cannot replicate in terminally differentiated cells but lytically infect dividing cells and this effective tumour targeting strategy has allowed development of HSV1716 as a potent oncolytic therapeutic agent. HSV1716 effectively kills tumour cell lines in tissue culture and, in a range of murine cancer models, the virus has induced tumour regression and increased survival times (Kesari et al 1995, MacKie et al 1996, Randazzo et al 1997). An excellent safety profile has been demonstrated in clinical trials following direct intratumoral injection of HSV1716 in patients with recurrent glioma (Rampling et al 2000, Papanastassiou et al; 2002, Harrow et al; 2004), metastatic melanoma (MacKie et al 2001) and squamous cell carcinoma of the head and neck (Mace et al unpublished). In each of these trials there was no evidence for spread of HSV1716 to surrounding normal tissue and the selectivity of the virus for replication in tumour cells alone has immense therapeutic potential for the treatment of many human malignancies. Currently, HSV1716 has been awarded orphan drug status for treatment of recurrent glioma and a Phase II/III clinical trial has recently been initiated.
The expression of exogenous genes such as enzymes for prodrug activation or transporters for the uptake of radioactive compounds will augment the oncolytic activity of HSV1716 by enhancing its ability to destroy tumour cells. Two such variants, HSV1716/NAT, which expresses the noradrenaline transporter (NAT) for the specific uptake of radiolabelled compounds such as [131I]MIBG, and HSV1790, that expresses nitroreductase, an enzyme capable of activating the prodrug CB1954, have respectively enhanced glioma cell cytotoxicity in tissue culture (Quigg et al, 2005). HSV1716/NAT and HSV1790 were generated by homologous recombination using an RL-1 shuttle vector that contained the NAT/nitroreductase expression cassette inserted within the ICP34.5 deleted region; cotransfection of BHK cells with the shuttle plasmid and HSV-1 strain 17+ DNA resulted in homologous recombination at the RL-1 loci with the resultant virus possessing an HSV1716 backbone with a NAT/nitroreductase expression cassette within the ICP34.5 deletion. However, homologous recombination is relatively inefficient with recombinant viruses produced in low numbers and isolation requires many rounds of time-consuming plaque purification to remove residual wild-type virus. For the development of an accelerated vector programme, which will allow production of large numbers of different second generation HSV1716 variants to be screened for enhanced tumour destruction, it will be advantageous to create recombinant viruses more rapidly and efficiently.
HSV1790 (also called HSV1716/CMV-NTR/GFP) is described in WO 2005/049845 and has been deposited in the name of Crusade Laboratories Limited having an address at Department of Neurology Southern General Hospital 1345 Govan Road Govan Glasgow G51 5TF Scotland on 5 Nov. 2003 at the European Collection of Cell Cultures (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession number 03110501 in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (herein referred to as the ‘Budapest Treaty’).